The present invention relates to test methods that can be used for clinical and non-clinical fundamental research and/or other studies to determine the consequences of a challenge to a responsive system, such as to determine the efficacy of an action, substance and/or product to prevent, induce, reduce or heal disease or injury to a responsive system. By using germane challenges to the specific responsive systems, the present invention of using a secondary challenge to enhance and/or prolong the effects of a primary challenge is amenable for use with challenges to multi-cellular and unicellular organisms such as animals, plant, fungi, bacteria, and the like, to organs of multi-cellular organisms such as epidermis, liver, heart and the like, and to tissues or individual cells or cell cultures such as isolated cells from multi-cellular organisms such as sperm, eggs, white blood cells, neurons, and the like or cultures of such cells. One embodiment of the present invention enhances and/or prolongs the effects of a primary challenge to human epidermis by using a different secondary challenge.
Studies on the effects of a challenge (insult) to provoke a human physiological response are needed to understand the etiology of disease and to develop both preventative measures and efficacious treatments. For example, studying the effects of stool on the etiology of diaper rash is needed to develop and/or determine efficacy of products, medications and/or processes that effectively prevent and/or treat diaper rash, which continues to routinely occur among many diapered children. Despite the many studies done to date, the etiology of diaper rash and the mechanisms of various diaper rash preventative and curative agents is incompletely understood. In general, this can be said for most dermatological diseases, as well as many common diseases of other tissues, organs and organisms.
Clinical and non-clinical scientific studies of biological responses, such as of diaper rash and many other dermatological responses, are hindered when current methods and/or devices cannot detect weak responses and/or distinguish among multiple causes for an observed response (i.e., disentangle or interpret a confounded response). Such studies are further hindered when the response being studied, such as diaper rash, occurs with relatively low and unpredictable frequency among the general population.
A common procedure to enhance a weak or inconsistent response is to damage or prime the responsive system prior to challenging it, but the enhanced response is often confounded. For example, the scarification test (Frosch and Kligman, 1976, Contact-Dermatitis 2: 314-242) has long been used to study irritant responses by first physically scarring skin with a needle prior to challenging that damaged skin with substances suspected to have mild irritancy or a weak response. Studies of allergenic and sensitization responses of human skin have long applied this approach by first priming the responsive system with repeated challenges, often over several weeks, to study effects of a second challenge of the same or different type (e.g., Kaidbey and Kligman, 1980, Contact Dermatitis 6:161-169).
There are two concerns with first damaging or priming a responsive system to study responses of that system. First, the damage or modification to the responsive system can cause the responsive system to respond to a challenge by a different mechanism than would the same naxc3xafve (undamaged and/or unmodified) responsive system. Second, the degree or severity of the response to a second challenge by a responsive system that has already been damaged or modified can be completely erroneous compared to a naxc3xafve responsive system. For example, a second challenge to already damaged skin could result in a severe response when the response by naxc3xafve skin would have been nil. Moreover, when the subjects are infants there is heightened justifiable concern anytime a severe response is observed and researchers would rather avoid inducing severe responses by any available means.
A common procedure to minimize confounding of a response, particularly when trying to study mechanisms of disease progression and/or the effects of interventions on the disease or on a injury, is to control the challenge, such as by using direct patching methods in dermatology studies. Such methods, however, are often inadequate when the response occurs at low frequency in the study population and/or the tools used to assess the response are relatively insensitive. To overcome these problems, a direct patching method is often used to study dermatological responses. However, these methods, when they work, typically require lengthy and/or repeated exposures that can be excessive and/or irrelevant to the normal course of events. In addition, many researchers and/or subjects find current patching methods sufficiently inconvenient and costly in time and resources that development of abbreviated methods is desirable.
The present invention is a novel approach to study physiological responses, particularly where enhancing and/or prolonging a response to a challenge without changing the inherent nature of the response is desirable. The methods are useful when the response of interest needs to be studied under both highly controlled and poorly-controlled challenge scenarios. In addition, the present invention reduces the challenge duration needed to provoke an enhanced and/or prolonged response. A specific embodiment of the present invention accomplishes this by enhancing and/or prolonging the effects of the primary challenge (stool) to human epidermis by using a different secondary challenge (tape-stripping) to elicit enhanced and durable erythemic and increased transepidermal water loss (TEWL) responses. In addition, the response to the initial challenge typically corresponds closely to what normally occurs with subjects during their normal lives because the exposure duration is relevant to the common experience of the subjects. Moreover, the primary challenge is applied to healthy, non-damaged skin so that the response is not confounded with the skin""s responses to any other pre-damage challenge.
The present invention relates to a test method to enhance and/or prolong the response of a primary challenge to a responsive system, so that the primary challenge can be better studied, by using a different secondary challenge that does not alter the mechanism by which the primary challenge affects the responsive system nor does it confound the response of the primary challenge. Further, the present invention typically: 1) reduces the exposure duration needed for the primary challenge to induce a response to one that is more relevant to normal exposure scenario(s) for the responsive system or subject, 2) reduces the number of subjects needed to observe statistically significant and/or clinically relevant effects, 3) is more convenient for the researchers, and 4) is minimally stressful to the responsive system or subjects. In addition, the primary and/or secondary challenge may be preceded by one or more interventions in studies designed to understand the etiology of the disease and/or determine the efficacy of a preventative measure. In addition, the secondary challenge may be preceded by and/or followed by one or more interventions in studies designed to determine the efficacy of a curative measure.
As used herein, xe2x80x9cstudyxe2x80x9d refers to either clinical or non-clinical research with specified testable hypotheses. As used herein, xe2x80x9csubjectxe2x80x9d refers to a human, lower animal species, or other specified organism participating in a study. As used herein, xe2x80x9cresponsive systemxe2x80x9d refers to any organism, organ, tissue, mass of cells, or individual cell(s) for which a change in activity, appearance, function, structure, or other condition can be provoked or induced. As used herein, xe2x80x9cchallengexe2x80x9d refers to the entity and/or process of provoking, moderating or otherwise influencing a physiological (including biochemical) activity by exposure under defined conditions to one or more physical, chemical or biological conditions or actions. As used herein, xe2x80x9ctest sitexe2x80x9d refers to any physical location on the responsive system, entity or organism that is monitored during the course of a study. As used herein, xe2x80x9cprimary challengexe2x80x9d refers to the first challenge applied to one or more test sites, that elicits one or more response(s) (which may or may not be detectable until after the secondary challenge). The primary challenge is the main challenge of interest in the study. There may be more than one component to primary challenge (e.g., there may be a chemical challenge and a physical challenge or there may be two successive identical or different chemical challenges) wherein all components of the primary challenge precede the secondary challenge. As used herein, xe2x80x9csecondary challengexe2x80x9d refers to a challenge applied after the primary challenge to one or more test sites designed or intended to enhance and/or prolong the response of the primary challenge. Importantly, a secondary challenge does not confound the response of the primary challenge nor does it alter the mechanism by which the primary challenge elicits a response. As used herein, xe2x80x9cconfoundxe2x80x9d means to confuse the meaning or interpretation of an observation even in the presence of controls. As used herein, xe2x80x9cenhancesxe2x80x9d refers to increasing the magnitude or intensity of an entity or event, such as increasing the color intensity or area affected during a dermatological response to a challenge. As used herein, xe2x80x9cprolongsxe2x80x9d refers to increasing the duration of an event. As used herein, xe2x80x9crecovery periodxe2x80x9d refers to a defined period of time during which the responsive system has opportunity to overcome the effects of a challenge. As used herein, xe2x80x9cinterventionxe2x80x9d refers to any process, act, condition, substance and/or entity attempting to affect the outcome of a challenge, such as to prevent, reverse, and/or attenuate the response of a responsive system to a primary challenge. As used herein, xe2x80x9cpre-challenge interventionxe2x80x9d refers to any process of exposing a test site and/or control site to one or more physical, chemical or biological conditions prior to exposure to at least one primary challenge. Thus, where there are a series of primary challenges, a pre-challenge intervention can occur anytime so long as it precedes one or more of the primary challenges. As used herein, xe2x80x9cpost-challenge interventionxe2x80x9d refers to any process of exposing a test site and/or control site to one or more physical, chemical or biological conditions subsequent to exposure to any primary challenges. The xe2x80x9cpost-challenge interventionxe2x80x9d can precede one or more secondary challenges, occur between/among one or more secondary challenges, or can follow one or more secondary challenges. As used herein, xe2x80x9cconcurrent-challenge interventionxe2x80x9d refers to any process of exposing a test site and/or control site to one or more physical, chemical or biological conditions during one, or more, of the primary challenger(s). As used herein, xe2x80x9cresponsexe2x80x9d refers to any detectable condition or change in condition of a responsive system. For example, a change in visual rash severity or TEWL before and after a challenge is a measure of response to that challenge. The method of detection includes but is not limited to methods utilizing human skills only and/or chemical, physical, mechanical, and/or electrical methods and equipment. As used herein, xe2x80x9cassessingxe2x80x9d refers to any method of evaluating, observing, noting, and/or comparing the condition (e.g., response) of a test site. As used herein, xe2x80x9cpate test gradexe2x80x9d refers to a visible skin response scored using a standardized scale (see table 17, page 99 in McNamara, 1976, in xe2x80x9cNew Concepts in Safety Evaluationxe2x80x9d, a modification of which is incorporated herein with detailed text). As used herein, xe2x80x9cdiaper rashxe2x80x9d refers to a response indicated by the occurrence on diapered epidermis of what is clinically defined as either erythema or dermatitis, or both, where persistent redness, dryness, pustules and/or other symptoms associated with such conditions may arise. As used herein, xe2x80x9crashxe2x80x9d refers to a response indicated by the occurrence on epidermis of what is clinically defined as erythema, dermatitis, psoriasis, or any of several other skin conditions where persistent redness, dryness, papules, pustules and/or other symptoms associated with such conditions may arise. As used herein, xe2x80x9cTEWLxe2x80x9d refers to trans-epidermal water loss, which is a measure of water movement across and out of the epidermis (skin). TEWL is related to the integrity of skin barrier function when measured under standardized conditions of temperature and humidity after the subject has been sufficiently equilibrated to those standardized conditions. As used herein, xe2x80x9cstoolxe2x80x9d refers to feces from human intestines. As used herein, xe2x80x9cstool analogxe2x80x9d refers to blends of irritative components commonly found in stool including, but not limited to, SHIME, enzymes, bile acids, candida species and mixtures thereof. As used herein, xe2x80x9cSHIMExe2x80x9d (Simulation for the Human Intestinal Microbial Ecology) refers to a biologically-simulated human fecal material created in laboratory digestion chambers having a bacterial consortia and both physical and chemical conditions representative of the various segments of the human intestinal tract. SHIME is designed to have representative characteristics of human feces from healthy adults or infants fed a varied diet, but lacks any pathogenic microbes to avoid the spread of disease and lacks any substantive amount of bulking or solidifying components, like cellulose. SHIME is further described in the following two references which are incorporated herein by reference in their entirety: 1. Molly, K., et. al., 1993. Development of a 5-step multi-chamber reactor as a simulation of the human intestinal microbial ecosystem. Appl. Microbiol. Biotechnol. 39:254-263; and 2. De Boever, P., et. al., 200X (in review). Development of a six-stage culture system for simulating the gastrointestinal microbiota of infants. Microb. Ecol. Health Dis. As used herein, xe2x80x9ctape-strippingxe2x80x9d refers to the act of applying a material of known adhesive properties to skin in a prescribed manner and removing that material for the purposes of subjecting the skin to a physical challenge. Materials of known adhesive properties useful for tape-stripping, which need not specifically be in a tape format, include adhesive tapes such as D-squame(copyright) and Sebutape(copyright) (CuDerm Corporation, Dallas, Tex.) or Blenderm(trademark) and ScotchTape(trademark) (3M Company, St. Paul, Minn.), and hydrogels such as Hypan(copyright) (Hymedix International, Inc., Dayton, N.J.), and other types of materials with adhesive properties such as glues, gums, and resins. As used herein, xe2x80x9ccontrol sitexe2x80x9d or xe2x80x9ccontrolxe2x80x9d refers to any test site and/or study treatment used to establish a standard of comparison for judging experimental effects and/or the degree of variation in effects, such as those associated with experimental noise, that occur during a study. As used herein, xe2x80x9cchallenge(d) sitexe2x80x9d refers to any test site that receives a primary and subsequent secondary challenge. As used herein, xe2x80x9cnegative controlxe2x80x9d refers to any control site that is treated identically to a challenged site except it receives neither a primary nor a secondary challenge. For instance, where a responsive system is first cleansed with water and then subjected to a primary and secondary challenge at a given temperature, the control site would also be cleansed with water and would be subject to the same given temperature, but would not be subject to the primary nor secondary challenge. As used herein, xe2x80x9cprimary controlxe2x80x9d refers to a control site that receives the primary challenge, but no secondary challenge. A primary control may or may not receive a concurrent-challenge intervention, pre-challenge intervention, or post-challenge intervention. As used herein, xe2x80x9csecondary controlxe2x80x9d refers to a control site that receives the secondary challenge, but no primary challenge. Where beneficial to a study design, a secondary control may receive a post-challenge intervention, even though it has not received a primary challenge. As used herein, xe2x80x9cpositive controlxe2x80x9d refers to a control site that receives a substitute primary challenge known to induce a defined response with or without the secondary challenge. All percentages, ratios, and parts herein, in the Specification, Examples, and Claims are by weight unless otherwise stated. The term xe2x80x9cm2xe2x80x9d, as used herein, means square meters. The term xe2x80x9ccm2xe2x80x9d, as used herein, means square centimeters. The term xe2x80x9ccmxe2x80x9d, as used herein, means centimeters. The term xe2x80x9cgxe2x80x9d, as used herein, means gram. The term xe2x80x9cmlxe2x80x9d, as used herein, means milliliter. The term xe2x80x9chrxe2x80x9d, as used herein, means hour. The term xe2x80x9cRHxe2x80x9d, as used herein, means relative humidity. The term xe2x80x9cpsixe2x80x9d, as used herein, means pounds per square inch. The term xe2x80x9cwtxe2x80x9d, as used herein, means weight. As used herein, the term xe2x80x9ccomprisingxe2x80x9d means that the various components, ingredients, or steps, can be conjointly employed in practicing the present invention. Accordingly, the term xe2x80x9ccomprisingxe2x80x9d encompasses the more restrictive terms xe2x80x9cconsisting essentially ofxe2x80x9d and xe2x80x9cconsisting of.xe2x80x9d
A. General Method
The test method of the present invention enhances and/or prolongs one or more of the effects of a primary challenge to a responsive system. The method enables the primary challenge to be better studied by using a different secondary challenge that does not alter the mechanism by which the primary challenge affects the responsive system nor does it confound results of the primary challenge with other spurious responses.
More specifically, the method generally comprises assessing a test site of the responsive system in its pre-challenge condition; subjecting the test site to a primary challenge; preferably but not necessarily, assessing the test site for a response; subjecting the same test site to a secondary challenge, wherein the secondary challenge is designed to enhance and/or prolong a response of the responsive system to the primary challenge, without confounding the response nor altering the mechanism by which the primary challenge elicits a response; and assessing the response subsequent to the secondary challenge. Preferably, the response is assessed at least twice following the secondary challenge, wherein the first assessment is made soon after the secondary challenge, and the second assessment is made after a recovery period. The primary and secondary challenges of the present invention can comprise physical challenges, chemical challenges, and/or biological challenges. The primary and secondary challenges may comprise multiple or complex components (for example, a primary challenge comprising a poorly-defined extract or comprising chemicals A and B dissolved in a buffered matrix and heated to a specific temperature) and/or steps (for example, a primary challenge comprising a first chemical challenge concurrently with or followed shortly thereafter by a second chemical challenge (same or different from the first) to the same test site). However, preferably, but not necessarily, the primary and secondary challenges are single steps (or challenges) in the process. The methods claimed herein also preferably comprise the additional step(s) of subjecting test site(s) to one or more pre-challenge intervention(s), post-challenge intervention(s), and/or concurrent-challenge intervention(s). The introduction of these interventions is useful for determining the ability of the intervention to prevent, cure, heal, and/or reduce disease or injury to a responsive system.
The methods of the present invention typically reduces the exposure duration needed for the primary challenge to induce a response to one that is relevant to normal exposure scenario(s) for the responsive system or subject, and is both minimally stressful to the responsive system and/or subjects and convenient for researchers. As a result, the present invention often significantly shortens the duration of and/or number of subjects needed for studies. In addition, it may enhance a low-level response that can be more easily assessed with current measurement techniques.
Thus, the methods of the present invention are also useful in a study design, also claimed herein, wherein the study comprises the steps of the method as outlined above in addition to comprising the steps of creating one or more controls selected from the group consisting of negative controls, primary controls, secondary controls, positive controls, and mixtures thereof. The studies and/or methods of the present invention are preferably designed to understand of the etiology of a disease and/or the ability of an intervention or challenge to prevent, cure, heal, and/or reduce and/or induce damage or injury to a responsive system.
B. Responsive Systems and/or Subjects
The responsive systems and/or subjects utilized in the methods of the present invention include but are not limited to: multi-cellular and unicellular organisms such as humans, animals, plants, fungi, bacteria, and the like; organs of multicellular organisms such as epidermis, liver, heart and the like; and to tissues or individual cells or cell cultures such as those isolated cells from multi-cellular organisms including but not limited to sperm, eggs, white blood cells, neurons, stoma, and the like.
C. Challenges
The primary and secondary challenges used in the method defined above can be of a physical, chemical, or biological nature. Some examples of physical challenges include, but are not limited to: heat, cold, humidity, desiccation ultraviolet or other forms of radiant energy, dark, friction, abrasion, lubrication, electric or magnetic energy, pressure, vacuum, visual images, occlusion, and sound. Some examples of chemical challenges are any element of the periodic table or non-living compositions of those elements including, but not limited to: organic and inorganic acids and bases or conditions caused by their presence in aqueous systems, surfactants, metal and complexes thereof, salts, proteins, lipids, vitamins, polymers, pharmaceuticals, feces, urine, perspiration, hair, extracts, and/or enzyme digests. Some examples of biological challenges include, but arc not limited to: microbials, cells, viruses, bacteria, parasites, and/or other living entities (e.g., fungi known to cause dermatitis, amoeba or protozoa known to cause dysentery or other diseases, mosquitoes or mites known to directly induce a range of responses or indirectly induce a range of responses when they are vectors for the transmission of viruses or parasites).
The methods of the present invention include but are not limited to the embodiments listed below:
1. human skin briefly exposed to ultraviolet radiation and subsequent tape-stripping (physical-physical/primary-secondary challenge) where skin redness and/or TEWL is measured to evaluate the efficacy of a UV-blocking agent applied as a pre-challenge intervention;
2. human skin exposed to trypsin (enzyme) and subsequent tape-stripping (chemical-physical/primary-secondary challenge) where interleukin 1 alpha (IL-1xcex1) induction and/or TEWL is measured to evaluate the efficacy of an enzyme inhibitor applied as a pre-challenge intervention;
3. human skin exposed to Candida albicans (fungus) dosed into SHIME and subsequent tape-stripping (biological(and chemical)-physical/primary-secondary challenge) where visual dermatitis and/or TEWL is measured to evaluate the efficacy of a fungal inhibitor applied as a pre-challenge intervention;
4. human skin exposed to trypsin (enzyme) and subsequent acetone exposure (chemical-chemical/primary-secondary challenge) where visual erythema score and/or TEWL is measured to evaluate the efficacy of an enzyme inhibitor applied as a pre-challenge intervention;
5. bovine eye exposed to cosmetic product(s) and subsequent sodium lauryl sulfate exposure (chemical-chemical/primary-secondary challenge) where interleukin 1 alpha (IL-1 a) is measured to evaluate the irritancy of various cosmetic products;
6. ovarian carcinoma cells exposed to treatment chemical(s) and subsequent exposure to growth factors (chemical-biological(or chemical)/primary-secondary challenge) where cellular division is measured by microscopy.
The specific embodiments of combinations of responsive systems exposed to primary and secondary challenges and then measured for various responses are numerous and a complete listing of such combinations is impractical. It is understood that one of ordinary skill in the art will be able to apply the general method of the present invention to suit his/her needs in a wide variety of applications, given the guidance of the present specification and/or examples.
D. Biological and/or Chemical Primary Challenge/Tape-stripping Secondary Challenge
One preferred embodiment of the present invention, useful in studies aimed at quickly assessing the effect of a biological and/or chemical substance on a responsive system, such as skin or cell cultures derived therefrom, comprises the steps of: assessing a test site of a responsive system in its pre-challenge condition; subjecting the test site to a primary challenge, wherein the primary challenge comprises a biological and/or chemical challenge; subjecting the test site to a secondary challenge, wherein the secondary challenge comprises the physical challenge of tape-stripping; and assessing the response subsequent to the secondary challenge, preferably at least twice, wherein the first assessment is made soon after the secondary challenge and the second assessment is made after a recovery period. By tape-stripping subsequent to the primary challenge of interest, the response of the responsive system to the primary challenge is enhanced and/or prolonged. Additionally, typically the duration of the application of the primary challenge necessary to provoke a response is substantially reduced.
A preferred embodiment of the invention is a clinical or non-clinical study involving male and/or female subjects of any age during which normal; healthy test sites on each subject are identified, and from which selected challenge sites receive both a primary challenge for a defined period of time and a secondary challenge at a defined period of time after the primary challenge. A variety of appropriate control sites may be established. Some may receive the same primary challenge only (i.e., primary control), or the same secondary challenge only (i.e., secondary control). Others may receive neither a primary nor a secondary challenge (i.e., negative control). Some may receive an appropriate alternative primary challenge (i.e., positive control), which may or may not receive a secondary challenge. The test sites may also receive other types of control interventions as may be required for the interpretation of study results. In the preferred embodiment of the present invention, test sites may be assessed several times to record response(s) that may occur during the study, including, but not limited to the following times: 1) at basal (baseline) conditions prior to the primary challenge, 2) subsequent to the primary challenge, 3) subsequent to the secondary challenge, and 4) subsequent to a recovery period of defined duration after the primary challenge was initiated and subsequent to any secondary challenge.
When this preferred embodiment is used for studies to determine the efficacy of an action, process, substance and/or product (an xe2x80x9cinterventionxe2x80x9d) to prevent, induce, reduce or heal an impaired state or disease, the intervention is applied to appropriate test sites before, during, or after the primary or secondary challenge occurs at those test sites. More specifically, the primary and/or secondary challenge may be preceded by one or more pre-challenge or concurrent-challenge interventions in studies to understand the etiology of the impairment or disease and/or determine the efficacy of a preventative measure, or the secondary challenge may be followed by one or more post-challenge or concurrent-challenge interventions in studies to determine the efficacy of a curative measure.
This protocol is designed to study the etiology of diaper rash due to skin exposure to stool. It is understood that amendments to the test protocol may be appropriate to study related objectives. The protocol comprises three visits by subjects to a test facility where the study is conducted. The first visit comprises interviewing subjects for possible inclusion in the study, and providing to those subjects who will potentially participate any applicable training and/or materials needed to initiate participation. The first visit is followed by a wash-out period generally comprised of the potential subjects exposing those areas of their skin that are of interest to standardized conditions intended to help minimize experimental noise by eliminating some confounding prior exposures of those skin sites to variable substances, actions and/or environmental conditions. After the wash-out period, the second visit to the test facility comprises re-evaluation of the potential subjects by investigators for inclusion in the study and, if accepted into the study, skin test sites are identified on the subjects which receive challenges for which assessments are made both before and after those challenges to discern skin responses to the challenges or lack thereof. The third visit to the test facility is comprised of assessments made of the challenged skin of subjects to discern persistence of any skin response or lack thereof.
A more specific description of a preferred protocol follows:
First Visit: Subjects are chosen from among healthy infants aged 1 to 36 months, preferably aged 9 to 27 months, and their healthy biological mothers aged 18 to 50 years, preferably 21 to 35 years. Preferably all potential subjects chosen, both adult and child, meet the following inclusion criteria (it is understood that the criteria may change depending upon the study objectives): 1) healthy and evenly-colored skin at all potential test sites with an absence of blemishes, burns, lesions, moles, scars and the like; 2) good general health as determined by having no communicable diseases nor chronic skin or other disorders; 3) neither currently taking nor having taken within two weeks of study initiation any medication(s), either topical or systemic, which in the opinion of the study investigator(s) might influence the skin condition or cause skin rash, such as erythema or dermatitis, or might increase BM frequency (e.g., oral antibiotics, anti-fungal agents, antihistamines, corticosteroids); 4) exhibit no significant hypersensitivity, rash or other abnormal skin reactions or lesions to topical or systemic medications, sunscreens, cosmetics, lotions, creams or fragrances within one year prior to study initiation; and 5) have adequate rash-free area as evaluated by an expert skin grader (i.e., patch test grade is less than 1.0 on a 0 to 4.0 half-point scale) for test sites on the child""s buttocks and on the adult""s forearms. All potential infant subjects chosen meet the following criteria: 1) child wears disposable diapers full-time; 2) child has no regular occurrence of diarrhea and no diarrhea at least four days prior to nor during the study; and 3) child is preferably transitioning to or already eating table food, where supplementation with formula is acceptable if it constitutes 50% to 99%, preferably 25% to 49%, most preferably 0% to 24% of the diet by caloric intake and where use of table foods and/or prepared baby and toddler foods such as baby cereals and jarred foods comprise the remainder of the diet. All potential adult subjects chosen meet the following criteria: 1) parent is willing to use standard diapers and standard wipes provided on her child instead of their normal brand(s) of diapers and wipes for the duration of the study; 2) parent agrees to use no lotions, creams, ointments, powders, topical medications, or other skin preparations in the diapered area of her child nor any such substance on her own forearm(s) at least 24 hours prior to the first challenge and through to the end of the study; 3) parent agrees to not bath her child""s diapered skin area nor bath her own forearms between the primary and secondary challenge of the study, nor bath any such skin areas during the recovery period; 4) parent agrees not to allow her child to consume nor to consume herself any food or beverage containing caffeine at least four hours before any clinical measurement(s) are made; 5) parent agrees not to use nicotine containing products at least 2 hours before clinical measurement(s) are made; and 6) parent must be willing to have her child""s buttocks and her own forearm(s) patched with the child""s own stool and subsequently have some skin sites tape-stripped.
It is generally understood that only the infant subjects can be studied or only the adult subjects can be studied by making appropriate changes to this general protocol. For example, if only infant subjects are studied, then adults need only participate to the extent necessary to enable study of their children.
Wash-out period: All subjects that meet inclusion criteria during an initial interview are provided with instructions to begin a wash-out period of 24 to 336 hours (1 to 14 days), preferably 72 to 96 hours (3 to 4 days), during which time the child wears exclusively the standard diapers and the parent uses exclusively the standard wipes to clean the child""s diapered skin during all diaper changes. Child and adult subjects typically wear their normal clothing and use their normal bathing practices including those soaps, shampoos and the like normally used. During wash-out subjects are not to apply any lotions, creams, ointments, powders, topical medications, and the like on potential test sites 6 to 168 hours prior, preferably 24 to 48 hours prior to the second visit to the test facility. In addition, all subjects are to bathe normally all potential test sites (i.e., baby diapered skin and adult forearms) 0.5 to 6 hours prior, preferably 1 to 2 hours prior to the second visit to the test facility. In addition, it is generally understood that the conditions of the wash-out period are defined by the needs of the study design such that modifications to the methods and materials given herein are acceptable provided they are recorded with a rationale.
Second visit: Subjects report to the test facility for their second visit at pre-determined appointments and the following procedures are performed:
1. Subjects are re-evaluated for suitability for patching against the inclusion criteria and for compliance with instructions for the washout period.
2. Typically, four healthy, unblemished, non-erythemic, non-dermatitic, non-overlapping skin sites approximately 5 cm by 5 cm in size or larger are identified on each infant""s buttocks and on one, or both, volar forearm(s) of each mother for use in the study, where the skin sites on each infant are preferably 2 sites located on contralateral (apposing) buttocks and on each mother""s forearm are 4 sites located linearly between the wrist and inner elbow. Fewer or additional sites and/or alternative site locations may be chosen as needed to meet the study objectives. The corners of each identified skin site are typically marked using non-toxic, water-soluble marking pens so they can be found during the course of the study.
3. Subjects are sent to a climate-controlled room having a standard temperature (typically 20 to 25xc2x0 C.) and humidity (typically 40%xc2x15% RH). Subjects"" skin sites are typically equilibrated to reach their basal (baseline) conditions by completely exposing their skin sites for 20 to 30 minutes. Longer equilibration time may be used if standardized for all subjects, but shorter equilibration time is often insufficient for most subjects"" skin to reach basal conditions.
4. After equilibration, assessments are made in the climate-controlled room at each test site to establish basal conditions of the test sites. Typically assessments are made for erythema by an expert skin grader using a patch grade scoring system (0 to 4.0 half-point scale) and for TEWL using a DermaLab(copyright) Evaporimeter with TEWL module (Cortex Technology, Hadsund, Denmark) that measures evaporation rate (g water/m2/hr) and computerized software for automated data collection and analyses (cyberDERM, inc., Media, Pa.). The general procedure for their use is as follows: the evaporimeter probe is applied to each skin site using a standard ring weight that applies equal pressure to all sites and TEWL data are collected for 60 seconds using automatic data collection by a computer linked to the evaporimeter, and where only the final 40 seconds of data are used to calculate a mean TEWL value. Details of operation for both the evaporimeter and software are available in their respective users manuals, which are incorporated herein by reference. In addition, a clean disposable evaporimeter probe cover is used to measure each site to avoid cross-contamination of sites with any challenges and/or interventions applied.
5. The test sites of each subject are randomly assigned to interventions for any combination of challenges, interventions and control conditions to meet the needs of the study design.
6. Stool is sampled from subjects, preferably from infant subjects, 48 to 168 hours, preferably 2 to 48 hours, more preferably 0.5 to 2 hours prior patching the primary challenge during to the second visit. Stool should be collected generally as soon after defecation as possible, such as within 60 minutes, preferably within 15 minutes. Stool can be sampled in various ways such as directly from the perianal grove of the child, from stool in a diaper that is uncontaminated by the diaper materials, or by any other appropriate means that are convenient and pose no harm to the subject. Stool should be sampled with a sterile implement, such as a wood or plastic cervical scraper or popsicle stick, and placed into sample containers, such as clean glass, polyethylene or polypropylene vials with lids. Stool analog such as a synthetic stool, such as SHIME, which is known to be free of pathogenic species, is a possible substitute primary challenge material. Once sampled, stool or SHIME for patching should preferably be stored at approximately 4xc2x0 C. until needed for preparing patches. Such storage should not exceed 168 hours, preferably not more than 48 hours, more preferably not more than 4 hours prior to patching, most preferably stored and used in patching within 1 hour after defecation.
7. Primary challenge patches are then prepared for subjects. It is preferred that infant subjects are patched with their own stool and parent subjects are patched with their child""s stool. The patches are preferably prepared prior to or concurrently with the assignment of skin sites to treatments (although patches could be prepared immediately and stored relative to the guidelines listed above for storing stool). An occlusive square (25.8 cm2) patch is recommended, such as a Hilltop Chamber Systems Patch (a.k.a. Hilltop Chamber Patch, Hilltop Research, Cincinnati, Ohio) consisting of a 2.5 cm diameter nonwoven cotton pad (Webril(copyright)) contained within a plastic chamber (e.g., Hilltop Chamber) covered by and held securely to the skin on all sides with an occlusive, hypoallergenic tape (e.g., Durapore(copyright)). Non-irritating, hypo-allergenic, occlusive patches made of natural and/or synthetic fibers like as cellulose, rayon or polyolefin may be acceptable alternatives, such as The Former Professional Medical Patch Pad (Kendall Company, Mansfield, Mass.) square of 14.5 cm2 consisting of a nonwoven cotton pad (e.g., Webril(copyright)) square of 3.6 cm2 covered by and held securely to skin on all sides with an occlusive, hypoallergenic tape (e.g., Blenderm(trademark), 3M Company, St. Paul, Minn.). Typically patches containing between 0.01 g and 1.0 g per cm2 skin surface, more preferably between 0.25 g and 0.35 g per cm2 skin surface of the stool are sufficient to elicit a desired response. However, it is understood that applying less stool (or less of some other primary challenge) is possible, but this may increase the exposure time necessary to elicit the desired response. Similarly, applying more stool is possible (or more of some other primary challenge) is possible, but this may not be appropriate, practical, economical or pleasant for the subject. Therefore, the amount of stool is not deemed a critical nor limiting aspect of the present invention. The patches may be wetted with between 0.1 g and 1.0 g of distilled and/or deionized water per cm2 skin surface, preferably where the patches may be wetted with between 0.01 g and 1.0 g of synthetic urine per cm2 skin surface, more preferably where the patches may be wetted with between 0.01 g and 1.0 g of natural urine per cm2 skin surface, and most preferably where the patches are unwetted except for liquid associated with the stool applied.
8. Subjects are patched for the primary challenge period for 0.5 to 16 hours, preferably for 1 to 8 hours, more preferably for 3.5 to 4.5 hours, such that the appropriate test sites receive the appropriate randomized interventions assigned in step 5. It is preferred that a standard cleaning method be used prior to applying patches to the skin test sites on child""s buttocks and parent""s forearm(s), as well as subsequent to removing all patches irrespective of treatment received. A recommended method is to use the standard wipes such that each individual skin test site is wiped 5 times where each time is a single stroke across only that site with the new standard wipe folded in thirds. Once patched, infants may be diapered and all subjects are free to engage in normal activities such as eating, sleeping, playing, etc., provided that the subjects remain at the test facility and avoid strenuous exercise or other activities that might cause perspiration to dislodge patches. In addition, infant subjects will have their diapers changed whenever soiled with stool as well as routinely during the primary challenge, preferably every hour, to avoid moisture accumulation under the patches.
9. Patches from the primary challenge period are removed and each patch site is cleaned using the same standard cleaning method (see 8 above). Subjects"" skin is equilibrated (as in 3 above) and assessments of any responses are made (as in 4 above).
10. While subjects are still in the climate control room a secondary challenge of five successive tape-strippings, using five different tapes, is made on test sites receiving the secondary challenge. Preferably the tape is left on for about 1 minute before removal. Tape stripping is preferably performed with D-Square tape (Cu-Derm, Dallas, Tex.), but alternative tapes, such as Blenderm(trademark) (3M Company, St. Paul, Minn.) or equivalent, or other appropriate adhesive materials may be substituted.
11. Tape-stripped test sites are allowed to re-equilibrate for about the same amount of time among all sites and among all subjects, preferably for 10xc2x12 minutes (not to vary by more than two minutes between sites within and among subjects) and assessments of any responses are made (as in 4 above).
12. Subjects leave the test facility and return the next day, preferably 24xc2x12 hours after the primary challenge was initiated. Subjects should not bath nor apply any substances (see inclusion criteria) to the test sites or neighboring skin until the third visit is complete.
13. During the third visit subjects"" skin is equilibrated (as in 3 above) and the final assessments of any responses are made (as in 4 above).